ABSTRACT Inflammatory microglia are a critical regulator of neuroinflammation and are strongly linked to Alzheimer?s Disease (AD). As microglia are now a lead target for therapeutic intervention in AD, there is a clear need to develop robust pre-clinical animal models that permit investigation of human microglia in vivo, which will enable the development of new microglia-based treatments for AD. This supplemental proposal is in response to NOT-AG-20-008. The goal of this supplemental proposal is to develop novel immunodeficient mice as a resource for the study of human microglia in AD. We have formed a multi-disciplinary team consisting of expertise in microglia and neurodegenerative diseases (Dr. Schafer) and expertise in creating, validating, and sharing new models of humanized mice with the scientific community (Drs. Shultz, Brehm, Greiner). We request supplemental funds to our ongoing R24 OD026440 project. The goal of the R24 project is to develop a resource of immunodeficient mice for the study of human stem cell-derived beta cells and muscle cells. This supplemental request will accomplish our goal of moving our analyses of human stem cells and their progeny into the setting of the brain, microglia, and AD. Human CSF1 and IL34 are cytokines required for microglial development as well as survival and support different subsets of microglia. The function and relationship of each subset to AD is not known. Our Scientific Premise is that our next generation NSG mice transplanted with human hematopoietic stem cells (HSC) will develop subpopulations of human microglia that will allow the investigation of different microglial subsets and their role in AD-related neurodegeneration. We have developed NOD-scid IL2rgnull (NSG) mice that transgenically express human CSF1 or human IL34. This will allow study of human microglia in a homeostatic state. We have also obtained a NSG-Tg(APP/PS1) strain from Dr. Howell at The Jackson Laboratory (JAX) that transgenically expresses a chimeric mouse/human precursor protein of A? and develops amyloid plaques. We will create NSG-Tg(APP/PS1 hIL34) and NSG-Tg(APP/PS1 hCSF1) mice to study microglia in AD. In Aim 1, we will validate and optimize the engraftment of human microglia in human HSC- engrafted NSG-Tg(hIL34), NSG-Tg(hCSF1), and in NSG-Tg(APP/PS1) mice expressing hIL34 or hCSF1. In Aim 2, we will define human microglia localization and immunological profile and determine their phagocytic and inflammatory properties basally and in the context of neurodegeneration associated with AD. As requested in NOT-AG-20-008, as a resource for our future investigation of human microglia in AD we will develop NSG-Tg(hIL34 hCSF1) mice and cross these mice with NSG-Tg(APP/PS1) mice to allow the study of both subsets of human microglia in a single recipient in a homeostatic and in a disease state, respectively. This will provide a critical resource of much needed validated platforms for investigating human microglia and their function in AD that will be readily available to the scientific community through the JAX Biorepository.